DESCRIPTION: The overall goals of this project are to refine and optimize a powerful new approach that we developed for the fabrication of high density RNA arrays and to demonstrate its utility in three important and interesting applications. These arrays are RNA analogs to the high density DNA arrays that have proven over the past two decades to be tremendously powerful tools for biomolecular analysis, particularly in the areas of global transcriptomics and genome-wide genetic variation analysis. In our approach, a high density DNA array is fabricated by standard photolithographic methods using photoprotected nucleoside phosphoramidites, the surface-bound DNA molecules are enzymatically copied into their RNA complements (also tethered to the surface), and the DNA templates are enzymatically destroyed, leaving behind the desired RNA array. Our strategy begins with technology development and optimization. The utility of RNA arrays depends strongly upon the array quality. Two key interrelated quality parameters are the RNA strand sequence fidelity and the RNA lengths that can be obtained. Other parameters of interest are the surface density of the RNA strands, the nature of the substrates employed for array fabrication, and the ability to employ modified nucleosides of various types. Tools needed to measure these parameters will be developed and used to guide optimization (maximize sequence fidelity and length) of the chemistry and enzymology employed for array fabrication. Other key facets of optimization include strand density, substrate types, and inclusion of various modified nucleosides. Technology development and optimization will be performed in the context of three separate applications that open new possibilities in biomolecular analysis: 1.) Identification and characterization of fluorescent RNA mimics of GFP; 2.) Determination of microRNA binding sites in 3'- and 5'- UTR mRNA sequences; and 3.) Determining the binding specificity of RNA-binding proteins. Taken together, the above program of targeted optimization and application of RNA array technology will yield a powerful and versatile new tool for biomolecular analysis that will open many new frontiers for research in the study of normal and disease biology.